Abstract

Efficacy
of chemotherapy is limited in numerous tumors by specific cellular
mechanisms that inactivate cytotoxic antitumoral drugs, such as
ATP-dependent drug efflux and/or drug detoxification by glutathione. In
reducing ATP pools and/or glutathione synthesis, it might be possible
to enhance the efficacy of drugs affected by such resistance
mechanisms. Reduction of the ATP pool and glutathione content is
achievable in cancer cells by depleting the exogenous methionine (Met)
supply and ethionine. Thus, the rationale for the present study was to
use Met depletion to decrease the ATP and glutathione pools so as to
sensitize tumors refractory to cytotoxic anticancer drugs. Met
depletion was achieved by feeding mice a methionine-free diet
supplemented with homocysteine. The effects of Met depletion combined
with ethionine and/or chemotherapeutic agents were studied using human
solid cancers xenografted into nude mice. TC71-MA (a colon cancer)
SCLC6 (a small cell lung cancer), and SNB19 (a glioma) were found to be
refractory to cisplatin, doxorubicin, and carmustine, respectively.
These three drugs are used to treat such tumors and are dependent for
their activity on the lack of cellular ATP- or glutathione-dependent
mechanisms of resistance. TC71-MA, SCLC6, and SNB19 were Met dependent
because their proliferation in vitro and growth in
vivo were reduced by Met depletion. Cisplatin was inactive in the
treatment of TC71-MA colon cancer, whereas a methionine-free diet,
alone or in combination with ethionine, prolonged the survival of mice
by 2-fold and 2.8-fold, respectively. When all three approaches were
combined, survival was prolonged by 3.3-fold. Doxorubicin did not
affect the growth of SCLC6, a MDR1-MRP-expressing tumor. A
Met-deprived diet and ethionine slightly decreased SCLC6 growth and, in
combination with doxorubicin, an inhibition of 51% was obtained, with
survival prolonged by 1.7-fold. Combined treatment produced greater
tumor growth inhibition (74%) in SCLC6-Dox, a SCLC6 tumor pretreated
with doxorubicin. Growth of SNB19 glioma was not inhibited by
carmustine, but when it was combined with Met depletion, survival
duration was prolonged by 2-fold, with a growth inhibition of 80%.
These results indicate the potential of Met depletion to enhance the
antitumoral effects of chemotherapeutic agents on drug-refractory
tumors.

INTRODUCTION

Metabolic anomalies are commonly found in solid tumors, and some
of them have been known for decades (1)
. Among the
metabolic abnormalities recurrently found in cancers,
Met3
dependency and
alterations of Met metabolism have been found in many, if not in all,
types of human tumors (2, 3, 4, 5, 6, 7, 8, 9, 10, 11)
. Met dependency leads to the
inability of cells to proliferate in culture when Met is absent and
replaced by Hcy, one of its metabolic precursors, whereas normal cells
grow in such a medium (2)
. This difference in the growth
of normal cells and Met-dependent tumor cells in
Met−Hcy+ medium might be due to the different
requirement for transmethylation reactions necessary to maintain their
proliferation rate. Methods for reducing in vivo Met intake
are to decrease the Met supply in food either using a Met-deprived diet
(6)
, a methioninase infusion (12, 13)
, or
Met-free total parenteral nutrition (14, 15)
. Met-depleted
diets effectively decreased metastatic potential and tumorogenicity in
an experimental rat model, as previously shown by us (16)
and by Guo et al.(8, 9)
.

In recent studies, we have obtained a potentiation of the antitumoral
effects of a Met-deprived diet by treating tumor-bearing mice
simultaneously with ethionine, a Met analogue. Ethionine might act by
inhibiting most methyltransferases and hence lead to DNA
hypomethylation, which can affect gene activity, as shown by Razin and
Riggs (17)
. Ethionine combined with a Met-deprived diet
can augment the effects of Met depletion alone in reducing the growth
of human prostate tumor (11)
and glioma (10)
xenografted into nude mice and of Yoshida sarcoma grafted into the rat
(9, 18)
. In vitro, we also observed that when
tumor cells were cultured in a Met−Hcy+ medium
containing ethionine, their ATP and glutathione pools were decreased,
and the cell cycle was blocked in S phase-G2, inducing an
irreversible arrest of DNA replication.

Current chemotherapy of cancer is based on the use of cytostatic and/or
cytotoxic agents acting on components and cellular functions that
control growth and cellular division. The failure of chemotherapy is
often due to direct or indirect target alterations that induce
resistance. Various parameters contribute to drug resistance, and some
of them are due to the increased expression of ATP-dependent mechanisms
such as P-glycoprotein (19, 20)
and MRP proteins
(21, 22)
. Drug resistance can also be related to a high
glutathione content and overexpression of some enzymes, such as
glutathione transferases (23, 24)
. Therefore, a decrease
of ATP or of the glutathione pools might counteract the ATP- or
glutathione-dependent mechanisms of resistance.

Experiments combining Met depletion with doxorubicin alone, doxorubicin
and vincristine, or 5-fluorouracil using Met-free parenteral nutrition
were conducted using the Yoshida sarcoma grafted into the rat
(14, 25, 26)
. They showed a potentiation of the
antitumoral effect of cytotoxic drugs. Similar assays were conducted
using a human gastric cancer grafted into nude mice, combining
Met-depletion with 5-fluorouracil (27)
. In patients with
gastrointestinal tract cancers, it was shown that Met depletion might
play a role as a modulator of 5-fluorouracil by decreasing the
free-thymidylate synthetase activity (27)
.

Met is an essential amino acid, and we have shown that Met depletion is
not compatible with long-term survival. Consequently, Met is
substituted by Hcy in the diet used in our experiments, thereby
allowing animals to survive (6)
.

In the present study, Met depletion was induced by a Met-deprived diet
and ethionine to decrease the cellular ATP and glutathione content and
to sensitize drug-resistant tumors to the effect of chemotherapeutic
drugs. This was tested using drug-resistant human tumors xenografted
into nude mice expressing Pgp and/or MRP, both ATP-dependent molecular
determinants of resistance, and/or a high level of glutathione. We have
treated xenografted human cancers, a glioblastoma, a small cell lung
cancer, and a colon cancer with a combination of a Met-deprived diet
and ethionine with carmustine, doxorubicin, and cisplatin, three drugs
used for therapy of such tumors.

MATERIALS AND METHODS

Cell Culture and Proliferation Assay

SNB19, TC71-MA, and SCLC6 were established as monolayer cell lines
as described previously (10, 11)
. For assays, cells were
maintained in RPMI 1640 (Sigma) supplemented with 10% FCS (Dutscher,
Brumath, France). Briefly, cells were plated in 10% FCS-RPMI 1640 for
24 h in a 5% CO2 atmosphere at 37°C at
105 cells/well in 24-well plates (ATGC, Noisy-le-Grand,
France) in triplicate. The medium was replaced by a Met-free medium
supplemented with dialyzed FCS, 100 μm folic acid, and
1.5 μm hydrocobalamin with 100 μm of Hcy
(Met−Hcy+) or Met
(Met+Hcy+), with or without 0.5 mg/ml ethionine
(all products from Sigma). Cell monolayers were fixed with methanol and
then stained with methylene blue. Methylene blue incorporated in fixed
cells was solubilized in hydrochloric acid (0.1 m), and the
absorbance of each well solution was measured (wavelength, 620 nm) with
a spectrophotometer (LP500; J Bio, les Ulis, France). The
cell proliferation index was calculated as the ratio of absorbances
corresponding to the assay medium to that of controls × 100.
Means and SDs were calculated, and statistical analyses were performed
with the Student’s t test.

Measurement of ATP Content

ATP content was quantified by a bioluminescence assay using a
Lumac biocounter M1500 (Lumac Perstop Analytical, Bezons, France), as
described previously (10)
. Cells were plated as described
above for 24 and 48 h, in Met−Hcy+ or
Met+Hcy+ medium with or without ethionine (0.5
mg/ml). Cell extracts were obtained using 1% trichloroacetic acid
(Sigma) in water. ATP was measured in a 450-μl mixture of
luciferin-luciferase (20 μl of a 40 mg/ml stock solution), ATP
standard (both from Sigma) (10 μl; 5.04 × 10−8m), and 10 μl of the sample to analyze in distilled
water. Results are expressed as percentages of ATP
nmol/10−6 treated cell extracts divided by ATP
nmol/10−6 control cell extracts. Experiments were repeated
five times. Means of data were calculated.

PCR Conditions.

The final PCR reaction volume was 50 μl. The cDNA solution (2.5 μl)
was pipetted into a sterile 0.2-ml tube, and the following mixture
containing 5 μl of 10× Taq buffer (Appligene-Oncor, Illkirch,
France), 1 μl of dNTP (final concentration, 2.5 mm each),
1 μl of each of the 5′ and 3′ primers (100 ng/μl), 38.5 μl of
water, and 0.5 μl (2.5 units) of Taq polymerase (Appligene-Oncor) was
added. After preheating at 94°C (hot start), the tubes were placed in
a Perkin-Elmer 2400 thermocycler (Yvelines, France) for 5 min at 94°C
followed by: (a) for the MDR-1 gene, 2 cycles (1
min at 94°C, 1 min at 62°C, and 1 min at 72°C) and 38 cycles (1
min at 94°C, 1 min at 60°C, and 1 min at 72°C), and a final
elongation at 72°C for 10 min; (b) for the MRP
gene, 35 cycles (1 min at 95°C, 1 min at 52°C, and 1.5 min at
72°C) and a final elongation at 72°C for 10 min; and (c)
for the β2 microglobulin gene, 3 min at 94°C, 35 cycles (1 min at
94°C, 30 s at 60°C, and 30 s at 72°C) and 10 min at
72°C.

Glutathione Assay

The glutathione assay was performed for the SNB19 (glioma)
xenograft in nude mice that were fed a
Met−Hcy+ diet combined with ethionine
treatment for 5 days. The tumor samples (50 mg) were pulverized with
20% salicylic acid and centrifuged. Tumor cells (106)
cultured 24 h in Met−Hcy+ medium without
ethionine, as detailed above, were similarly treated. For each assay,
500 μl of the supernatant were mixed with 500 μl of mixture buffer
(0.3 m Na2HPO4 (pH 7.5), 10
mm EDTA, and 0.2 mm DTNB) before reading
absorbance at 412 nm. The level of absorbance is proportional to the
glutathione pool and is expressed relative to the cultured cell number
(in vitro) or to the weight (in grams) of tissue (in
vivo).

In Vivo Studies

Mice.

Eight- to 10-week-old, Swiss nu/nu male or female mice
(20–25 g body weight) bred in the animal facilities of the Curie
Institute (Paris, France) were used in these assays. The animals were
maintained under specified pathogen-free conditions. Their care and
housing were in accordance with the institutional guidelines of the
French Ethical Committee (Ministère de l’Agriculture et de la
Forêt, Direction de la Santé et de la Protection Animale,
Paris, France) and supervised by authorized investigators.

Tumors and Xenotransplantation.

Xenografts were established by implantation of tumor samples into
the scapular area of the nude mice. These samples were taken either
from a tumor obtained previously by a s.c. injection of 10 ×
106 SNB19 glioma cells [obtained from Gross et
al.(30)
and subcultured in our laboratory] into the
flank of nude mice or from human tumor fragments obtained from the
clinical samples and established in serial transplantation, such as
TC71-MA, a colon cancer (31)
, and SCLC6, a SCLC
(32)
. Tumors were maintained by successive passages from
mouse to mouse. A SCLC6-Dox tumor derived from SCLC6 was established in
nude mice by i.p. injection of 30 mg/kg/body weight of doxorubicin
followed by transplantation 2 h later into a new nude mouse. This
procedure was repeated three times before use in the in vivo
assays (32)
.

Tumor Growth Inhibition Studies.

Mice were grafted with tumor fragments of approximately 15
mm3 in volume. Tumors appeared at the graft site 2–5 weeks
later. Mice bearing growing tumors with a volume of 60–100
mm3 were individually identified and randomly assigned to
the control or treatment group, and the treatment was started. The
animals bearing tumors were sacrificed when their tumor volume reached
2500 mm3, the level defined as ethical sacrifice. Volumes
of individual tumors were calculated from the measurements of two
perpendicular diameters using a caliper, performed every 2 days. Each
tumor volume (V) was calculated according to the following
formula (33, 34)
: V = a2 ×
b/2, where a and b are the smallest and
largest perpendicular tumor diameters. RTVs were calculated from the
formula: RTV =
(Vx/V1), where
Vx = the volume on day x, and
V1 is the tumor volume at the initiation of
therapy (day 1). Growth curves were obtained for each individual tumor
by plotting values of RTV against time (expressed as days after the
start of treatment). The antitumor activity was evaluated according to
three criteria: (a) the tumor growth inhibition, which was
calculated according to the following formula: 100 −
(RTVt/RTVc) × 100 where RTVt is the mean RTV of the treated group
and RTVc is the mean RTV of the control group at the time of optimal
response; (b) the tumor growth delay, calculated as the time
in days required for the tumors to reach a 15-fold increase in RTV,
corresponding to the survival time of treated mice; prolongation of
survival was calculated as the ratio between the survival time in the
treated group and that of controls; (c) the tumor doubling
time was calculated as the delay in days required to double an initial
tumor volume of 200 mm3 (size in exponential growth phase).

The statistical significance of the differences between the tumor
volumes reached in each group was calculated with the ANOVA test
(GraphPad InStat, San Diego, CA) and the Student’s t test.

Formulation and Administration of Diets and Drugs.

Mice were fed either a regular diet (UAR, Villemoisson, France) or a
Met-deprived Hcy-supplemented diet in which the proteins were replaced
by an amino acid mixture without Met
(Met−Hcy+; Refs. 10 and 11
). Hcy (Sigma,
Grenoble, France) was added at 0.4 g/100 g of diet. Ethionine (Sigma)
was chlorohydrated and solubilized extemporaneously in water at a
concentration of 25 mg/ml, and 0.2 ml (200 mg/kg) of this solution was
injected daily by the i.p. route. Cisplatin (Rhône-Poulenc-Rorer,
Vitry-sur Seine, France) was solubilized in water at a concentration of
0.15 mg/ml, and 0.2 ml (1 mg/kg) of this solution was injected by the
i.p. route for 5 consecutive days every 2 weeks.

Carmustine (Bristol Myers Squibb, Paris, France) was solubilized in
NaCl 0.9% at a concentration of 0.33 mg/ml, and 0.1 ml of this
solution (1 mg/kg) was injected by the i.p. route for 3 consecutive
days. Doxorubicin (Pharmacia, Saint-Quentin-en-Yvelines, France) was
solubilized extemporaneously in water at a concentration of 0.5 mg/ml,
and 0.25 ml (5 mg/kg) of this solution was injected by the i.p. route
once a week for 2 or 3 weeks, as specified in the text.

Combinations of Met Depletion with Anticancer Drugs.

For all combinations, mice were fed a Met-deprived diet
(Met−Hcy+) throughout the experiment. For
doxorubicin combined treatment, ethionine (200 mg/kg/day, diluted in
water) was administered i.p. on days 1 and 2; two h after the second
injection of ethionine, doxorubicin (5 mg/kg, diluted in water) was
injected i.p. This treatment was repeated weekly for 2 or 3 weeks, as
reported in Fig. 3⇓
. Different control groups were used, namely, mice
treated with Met−Hcy+ diet or ethionine alone
(i.e., Met depletion), doxorubicin alone, or 0.9% NaCl. For
cisplatin combined treatment, mice fed a
Met−Hcy+ diet and receiving ethionine daily
were treated with cisplatin (1 mg/kg, diluted in water) for 5
consecutive days/week, every 2 weeks. Four treatment cycles were
performed. Different control groups were used, namely, mice treated
with ethionine, a Met−Hcy+ diet, or cisplatin
alone, a combination of a Met−Hcy+ diet with
ethionine or cisplatin or with NaCl. For carmustine combined treatment,
mice fed either a Met-deprived diet (Met−Hcy+)
and ethionine daily or a regular diet were treated with carmustine (6 mg/kg in 0.9% NaCl) for 3 consecutive days/week for 3 weeks.
Different control groups were used, namely, mice treated only with
carmustine or with Met−Hcy+ diet-ethionine or
with NaCl.

RESULTS

Biological Characteristics of the Xenografted Human Tumors Used.

All tumors originated from patients before any treatment, and their
human origin was checked by karyotype analysis (data not shown).
MDR-1 and MRP mRNA were expressed in TC71-MA
tumor, a colon cancer, and in SCLC6 and SCLC6-Dox, both SCLCs, but not
in SNB19, a xenografted glioma, as shown by reverse transcription-PCR
(Fig. 1)⇓
. MDR-1 and
MRP mRNA expression was not significantly increased in
SCLC6-Dox, despite pretreatment with a high dosage of doxorubicin.

In Vitro and in Vivo Effects of Met
Depletion in Human Tumor Xenografts.

The in vitro proliferation rate of SNB19 cells decreased
after several hours of culture in Met-free Hcy-supplemented medium
(Fig. 2)⇓
. Addition of ethionine to the
culture medium reduced the cell proliferation rate of SNB19, and
inhibition of cell proliferation by ethionine was greater in the
absence of Met. Measurement of ATP pools, performed in parallel with
the proliferation assay, showed a drop in ATP after 48 h of
culture in Met-free medium and ethionine. Glutathione content was
decreased in cells after a 24-h culture in Met-free medium, with or
without ethionine, as shown in Fig. 2⇓
. When grafted into nude mice,
growth of SNB19 glioma was reduced by 26% as compared to the control
group (P > 0.05); no prolongation of survival was
observed (Table 1)⇓
. In vivo
Met depletion was obtained in nude mice bearing xenografts by feeding
them a Met-free diet for 3 weeks. Treatment started as soon as the
tumor size reached a volume of 60–100 mm3. Met depletion
with a Met-free diet and ethionine inhibited tumor growth by 53%
(P < 0.01) and induced a prolongation in survival of
16 days (1.6-fold; P < 0.05; greater than the control
group). This was associated with a reduction in the glutathione content
of 500 mm3 SNB19 xenografts after treatment with a Met-free
diet and ethionine. A 4-fold decrease in glutathione content was
observed after 5 days of treatment in comparison with untreated tumors
(Fig. 2)⇓
.

Effects of Met deprivation and ethionine on
cell proliferation, ATP pools, and glutathione content of SNB19, a
human glioma. Top, in vitro cell proliferation assay in two
different media with Hcy, with and without Met, with and without
ethionine, added at a concentration of 0.5 mg/ml. Middle,
relative content in ATP of SNB19 cultured in Met-containing medium
(left) or in Met-free medium (right), as a
function of time. Bottom, glutathione content in cells
(left) and in tumor tissue (right), in controls,
Met-free medium, or diet and ethionine.

In vitro and in vivo antitumoral
effects of Met-free medium or Met-free diets with and without ethionine
(Eth)

The in vitro proliferation rate of TC71-MA cells decreased
after several hours of culture in Met-free Hcy-supplemented medium
(Table 1)⇓
. Addition of ethionine to the culture medium did not affect
the proliferation rate of TC71-MA, which was already very low. Growth
of the TC71-MA colon cancer was inhibited (46%; P <
0.05), prolonging the survival of mice by 12 days, which
represents a 2.1-fold improvement over controls (Table 2)⇓
. The combination of a Met-free diet
with ethionine, leading to a more complete Met depletion, inhibited
tumor growth by 56% (P < 0.01), prolonging the
survival of mice by 2.8-fold. The doubling time of the tumor was 13 and
26 days after feeding mice with a Met-free diet, without or with
ethionine, respectively.

Antitumoral effects of cisplatin, Met-free diet,
and ethionine on the growth of TC71-MA, a human colon cancer grafted
into nude mice

The in vitro proliferation rate of SCLC6 cells decreased
after several hours of culture in Met-free Hcy-supplemented medium
(Table 1)⇓
. Addition of ethionine to the culture medium did not change
that of SCLC6 (Table 1)⇓
. Growth of both SCLC6 and SCLC6-Dox was not
significantly reduced by Met depletion (Table 1)⇓
, and administration of
ethionine had no effect on SCLC6-Dox, whereas it moderately slowed the
growth of SCLC6 (see Fig. 4⇓
).

Combination of Met Depletion with Cisplatin for the Treatment of
TC71-MA Xenograft, a Colon Cancer.

Cisplatin did not inhibit the growth of TC71-MA (Table 2⇓
and Fig. 3⇓
). Ethionine alone or combined with
cisplatin given to mice fed a regular diet had no effect. When
cisplatin was administered to mice fed a Met-free diet, the growth
inhibition was 56% (46% with the diet alone), and the survival was
prolonged by 2.8-fold (2.1-fold with the diet alone). A similar effect
was obtained in mice fed a Met-free diet and treated with ethionine
(Fig. 3)⇓
. A superior effect was obtained by treating the tumor-bearing
mice with the combination of a Met-free diet, ethionine, and cisplatin,
which resulted in a growth inhibition of 61% and prolonged survival by
3.3-fold, although this was only slightly different from the
antitumoral effects of the Met-free diet with ethionine or the Met-free
diet with cisplatin. It was clear that the Met-free diet enhanced the
antitumoral efficacy of the two other compounds used.

Combination of Met Depletion with Doxorubicin for the Treatment of
SCLC6 Xenograft, a Small Cell Lung Cancer.

SCLC6, a SCLC expressing Pgp, MRP, and glutathione, was
refractory to the effect of doxorubicin. SCLC6-Dox tumors, pretreated
with doxorubicin, expressed the same determinants of resistance and
were also resistant to doxorubicin when tumor-bearing mice were fed a
regular diet (Table 3⇓
and Fig. 4⇓
). Doxorubicin reduced the growth of
SCLC6 tumors when mice were fed a Met-free diet and received ethionine.
Indeed, a 51% growth inhibition (P < 0.01) was seen,
and survival of mice was prolonged by 1.7-fold, whereas SCLC6 growth
was slowed slightly by the diet plus ethionine. Similar observations
were made with SCLC6-Dox tumors; an antitumoral effect of doxorubicin
was obtained by the combination of doxorubicin, a Met-free diet, and
ethionine, leading to a growth inhibition of 74% (P <
0.01) and a prolonged survival (1.7-fold; Fig. 4⇓
). These results
were all the more striking with SCLC6-Dox because it was not inhibited
at all by the Met depletion.

Antitumoral effects of doxorubicin, Met-free
diet, and ethionine on the growth of SCLC6, a human SCLC established in
nude mice, and on the growth of SCLC6-Dox, derived from SCLC6 after
treatment with doxorubicin, as detailed in “Materials and Methods”

Combination of Depletion with Carmustine for the Treatment of SNB19
Xenograft, a Glioblastoma.

A limited antitumoral effect of carmustine alone was obtained in SNB19
glioma (30% tumor growth inhibition; Table 4⇓
and Fig. 5⇓
). The combination of carmustine with
the Met-deprived diet and ethionine enhanced the antitumoral effect of
the Met deprived diet-ethionine association (80% and 46% tumor growth
inhibition, respectively), leading to a prolongation of mice survival
of 2-fold in the group receiving the triple therapy.

Antitumoral effects of carmustine, Met-free
diet, and ethionine on the growth of SNB19, a human glioma established
in nude mice

DISCUSSION

Met depletion of cells induced in vitro by substitution
of Met by Hcy in the culture medium and the addition of ethionine
reduced cell proliferation of cancer lines and decreased ATP and
glutathione pools. ATP is required for the function of drug efflux
pumps, such as Pgp encoded by the MDR-1 gene and MRP, which
are involved in the resistance to numerous compounds including
doxorubicin. Glutathione and glutathione transferases, which are known
to be constantly elevated in tumors as compared to normal tissues,
detoxify xenobiotics including the drugs doxorubicin, cisplatin, and
carmustine used in this study. Met depletion might allow recovery of
the antitumoral efficacy of these cytotoxic agents in drug-refractory
tumors by decreasing their resistance potential. In vivo,
animals can be starved of Met by replacing the standard diet with a
Met-free regimen and simultaneous administration of ethionine. Our
results confirm this hypothesis.

We and others have previously described the Met dependency of tumor
cells (3, 11, 35, 36)
. This led us to design a therapeutic
approach using a Met-deprived diet and ethionine, a Met analogue, both
contributing to Met depletion in animals. The Met-deprived diet was
well tolerated by nude mice when Hcy was added to substitute for Met.
Hcy is a precursor in endogenous Met synthesis and is used efficiently
by normal cells to maintain their metabolism, but not by tumor cells.
Ethionine treatment, combined with a Met-deprived diet, was not toxic
up to a daily dose of 1 g/kg body weight. In all tumors tested from
various origins, tumor growth inhibition was observed when ethionine
treatment was associated with the Met-deprived diet (11)
,
reaching a value of 80% with significant tumor growth delay,
prolonging survival times by 2–3-fold in tumor-bearing animals. Such
an antitumoral effect was confirmed in the present study in nude mice
bearing TC71-MA, a human colon cancer, and SNB19, a glioblastoma.

Met deprivation acts by reducing proliferation of tumor cells, and this
inhibition is not counteracted by Hcy but is reinforced by ethionine.
Met depletion decreases the glutathione content, irreversibly blocks
cells in S phase and G2 of the cell cycle, and induces
apoptosis (10)
. Combined with ethionine, it induced a drop
in ATP pools. Decreases in glutathione induced in the absence of Met
have been described previously (37, 38)
. In the present
study, we show that the combination of a Met-deprived diet and
ethionine induced a depletion of the glutathione pool in tumors
xenografted into nude mice.

The fundamental basis of the antiproliferative effect of Met depletion
in tumor cells might be partly due to dNTP imbalance induced by folate
deviation toward endogenous Met synthesis, which is responsible for the
S-phase blockade. The intracellular Met concentration determines the
metabolic priority of folate (39, 40)
. Under normal
conditions, folates are used for endogenous Met synthesis and purine
and pyrimidine bases. Thymidine, which is essential for DNA reparation
and replication, is the methylated analogue of uracil. This methylation
reaction, which is catalyzed by thymidylate synthetase, specifically
requires 5,10-tetrahydrofolate as a methyl donor. When the regular diet
is replaced by a Met-deprived diet, folate is diverted away from DNA
synthesis to the resynthesis of Met. As a result, there is an imbalance
in the dNTP pool. This, in turn, is known to promote an accumulation of
DNA strand breaks (41)
, impair DNA repair
(42)
, and lead to apoptotic death (43, 44)
.
DNA strand breaks and replication arrest (45)
could also
be induced by ethionine treatment. Ethionine, the Met analogue used, is
metabolized to S-adenosylethionine and thus might transethylate DNA,
RNA, and phospholipids. The DNA ethylation by S-adenosylethionine could
prevent polymerase recognition of the ethylated cytosine and induce the
S-phase blockade by a replication arrest or DNA strand breaks. DNA
hypomethylation induced by Met depletion (46, 47)
can also
induce an accumulation of DNA strand breaks (41)
.

We decided to combine metabolism-targeted therapy and Met depletion,
which induces a decrease in glutathione and ATP content, with
chemotherapeutic agents, whose efficacy could inversely depend on the
expression of Pgp, MRP, or glutathione. The choice of the cytotoxic
agent to be combined with Met depletion was dependent on the type of
tumor. For SCLC6, a small cell lung cancer, the association of
doxorubicin with Met depletion was based on the drug-refractory
phenotype of SCLC6, which expresses Pgp and MRP proteins
(48)
. The activity of Pgp could be responsible for
chemoresistance, and we showed previously that it could be reversed by
verapamil (49)
. Expression of MRP and of glutathione
S-transferase π probably contributes to the resistance of SCLC6.
Doxorubicin is used for treating patients (50)
, even if it
is not the reference treatment of SCLC. SCLC6, a doxorubicin-resistant
tumor, was a suitable model to show the capacity of increasing the
sensitivity to doxorubicin by Met depletion. Indeed, cell
chemosensitivity to doxorubicin is dependent on its intracellular
accumulation, with doxorubicin resistance being induced by the
alteration of membrane transport such as ATP-dependent mechanisms of
efflux or by a high glutathione concentration (51)
. The
decrease of ATP and glutathione pools induced by Met depletion in the
presence of ethionine might lead to the recovery of doxorubicin
chemosensitivity. Both SCLCs (SCLC6 and SCLC6-Dox) were very resistant
to doxorubicin and expressed MDR1, MRP, and
GSTπ, yet they responded to the combination of doxorubicin with Met
depletion by a significant inhibition of tumor growth and prolonged
survival, whereas doxorubicin alone had little or no activity. The
results observed could also be explained by the DNA intercalation and
topoisomerase II inhibition of doxorubicin, which could potentiate the
effects of the hypomethylation induced by the Met depletion.

Cisplatin has been used in the treatment of colon cancer (52, 53)
. TC71-MA, a human colon cancer, was poorly sensitive to
cisplatin. Several arguments led us to combine cisplatin with Met
depletion. Cisplatin interferes with Met transport and acts as an
inhibitor of amino acid entry. This was demonstrated in brain tissue
(54)
, and this could contribute to augment Met depletion
in tumor tissue. Alternatively, cisplatin detoxification was found to
require glutathione, which forms complexes with this heavy metal
(55)
, and furthermore, efflux of glutathione-cisplatin
complexes is driven by MRP, an ATP-dependent transporter
(22)
that could be reduced by Met depletion. The TC71-MA
tumor displayed a high level of glutathione and expressed MRP, like the
majority of colon cancers, and this could explain the lack of efficacy
of cisplatin. The association of a Met-deprived diet with cisplatin
enhanced the antitumoral efficacy of cisplatin. We hypothesize that the
simultaneous administration of a Met-deprived diet decreases the
glutathione pool, thereby decreasing the formation of
cisplatin-glutathione complexes and hence sensitizing TC71-MA to
cisplatin.

Carmustine is the reference drug for the treatment of glioma (56, 57)
. However, its efficacy is limited, like all nitrosoureas, by
glutathione detoxification, and a relationship between the response of
tumors to carmustine and their glutathione content has been described
previously (58)
. We hypothesize that the effect of Met
depletion and ethionine in decreasing the amount of glutathione could
be responsible for the potentiation of the antitumoral efficacy of
carmustine observed in SNB19 xenografts.

In conclusion, the potentiation of the antitumoral effect of a
Met-deprived diet, ethionine, and chemotherapy could be explained, at
least in part, by a decrease in glutathione and available ATP pools
induced by Met depletion and ethionine administration, which in turn
diminished the resistance potential of cancer cells to the cytotoxic
agents. The three tumors are representative of solid tumors refractory
to conventional therapy, yet they displayed significant responses to
chemotherapy when combined with Met depletion.

Acknowledgments

We are grateful to V. Bordier and C. Alberti for excellent
technical assistance in animal experimentation. We thank Dr. S. Agrawal
for helpful critical review and reviewing the English used in this
report.

Footnotes

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